The present invention is concerned with cloning and expression of a novel mammalian receptor protein, designated herein GFRα-4 and in particular with an isolated nucleic acid sequence encoding the GFRα-4 protein, an expression vector comprising said nucleic acid sequence, a host cell transformed or transfected with said vector, isolated GFRα4 protein, compounds which act as agonists or antagonists in relation to GFRα-4 and methods of identifying them, together with pharmaceutical compositions comprising the isolated nucleic acid, the receptor protein or said agonist or antagonist.
Neurotrophic growth factors are involved in neuronal differentiation, development and maintenance. These proteins can prevent degeneration and promote survival of different types of neuronal cells and are thus potential therapeutic agents for neurodegenerative diseases. Glial cell-line derived neurotrophic factor (GDNF) was the first member of a growing subfamily of neurotrophic factors structurally distinct from the neurotrophins. GDNF is a distantly related member of the transforming growth factor β (TGF-β) superfamily of growth factors, characterized by a specific pattern of seven highly conserved cysteine residues within the amino acid sequence (Kingsley, 1994). GDNF was originally purified using an assay based on its ability to maintain the survival and function of embryonic ventral midbrain dopaminergic neurons in vitro (Lin et al., 1993). Other neuronal cell types in the central (CNS) or peripheral nervous systems (PNS) have been shown to be responsive to the survival effects of GDNF (Henderson et al., 1994, Buj-Bello et al., 1995, Mount et al., 1995, Oppenheim et al., 1995). GDNF is produced by cells in an inactive proform, which is cleaved specifically at a RXXR recognition site to produce active GDNF (Lin et al., 1993). In view of its effects on dopaminergic neurons, clinical trials have evaluated GDNF as a possible treatment for Parkinson's disease, a common neurodegenerative disorder characterized by the loss of a high percentage (up to 70%) of dopaminergic cells in the substantia nigra of the brain. Exogenous administration of GDNF has potent protective effects in animal models of Parkinson's disease (Henderson et al., 1994, Beck et al., 1995, Tomac et al., 1995, Yan et al., 1995, Gash et al., 1996, Choi-Lundberg et al., 1997, Bilang-Bleuel et al., 1997, Mandel et al., 1997).
Recently, three new members of the GDNF family of neurotrophic factors have been discovered. Neurturin (NTN) was purified from conditioned medium from Chinese hamster ovary (CHO) cells using an assay based on the ability to promote the survival of sympathetic neurons in culture (Kotzbauer et al., 1996). The mature neurturin protein is 57% similar to mature GDNF. Persephin (PSP) was discovered by degenerate primer PCR using genomic DNA. The mature protein, like mature GDNF, promotes the survival of ventral midbrain dopaminergic neurons and of motor neurons in culture (Milbrandt et al., 1998). The similarity of the mature persephin protein with mature GDNF and neurturin is ˜50%. Very recently, a fourth member has been cloned using genomic DNA information in the public EMBL database and has been named Enovin (EVN) (Masure et al., 1999) or Artemin (ARTN) (Baloh et al., 1998b). This factor is ±57% similar to NTN and PSP and acts primarily on peripheral neurons.
All four GDNF family members require a heterodimeric receptor complex in order to carry out downstream intracellular signal transduction. GDNF binds to the GDNF family receptor alpha 1 (GFRα-1; also termed GDNFRα, RETL1 or TrnR1; GFRα Nomenclature Committee, 1997) subunit, a glycosyl phosphatidyl inositol (GPI)-anchored membrane protein (Jing et al., 1996, Treanor et al., 1996, Sanicola et al., 1997). The GDNF/GFRα-1 complex subsequently binds to and activates the cRET proto-oncogene, a membrane bound tyrosine kinase (Durbec et al., 1996, Trupp et al., 1996), resulting in phosphorylation of tyrosine residues in cRET and subsequent activation of downstream signal transduction pathways (Worby et al., 1996). GFRα-2 (also termed RETL2, NTNR-α, GDNFR-β or TrnR2), which is similar to GFRα-1, has been identified by a number of different groups (Baloh et al., 1997, Sanicola et al., 1997, Klein et al., 1997, Buj-Bello et al., 1997, Suvanto et al., 1997). The human GFRα-1 and GFRα-2 receptor subunits are 49% identical and 63% similar by protein sequence with 30 of the 31 cysteine residues conserved. Both receptors contain a hydrophobic domain at their carboxy-termini involved in GPI anchoring to the membrane. GFRα-1 and GFRα-2 are widely expressed in almost all tissues and expression may be developmentally regulated (Sanicola et al., 1997, Widenfalk et al., 1997).
GFRα-1 is the preferred receptor for GDNF, whereas GFRα-2 preferentially binds neurturin (Jing et al., 1996, Treanor et al., 1996, Klein et al., 1997). It is also clear, however, that there is some cross-talk between these growth factors and receptors as GDNF can bind to GFRα-2 in the presence of cRET (Sanicola et al., 1997) and neurturin can bind to GFRα-1 with low affinity (Klein et al., 1997). GDNF and neurturin are thus part of a neurotrophic signalling system whereby different ligand-binding subunits (GFRα-1 and GFRα-2) can interact with the same tyrosine kinase subunit (cRET).
Recently, a third member of the GFRα family of coreceptors, GFRα-3, has been described (Jing et al., 1997, Masure et al., 1998, Worby et al., 1998, Naveilhan et al., 1998, Baloh et al., 1998a). This receptor's amino acid sequence is 35% identical to both GFRα-1 and GFRα-2. GFRα-3 is not expressed in the developing or adult CNS, but is highly expressed in several developing and adult sensory and sympathetic ganglia of the PNS (Widenfalk et al., 1998, Naveilhan et al., 1998, Baloh et al., 1998a). GFRα-3 has been shown to be the preferred coreceptor for Enovin/artemin and also signals via cRET (Masure et al., 1999, Baloh et al., 1998b). Crosstalk between EVN/ARTN and GFRα-1 seems also possible, at least in vitro.
A fourth member of the GFRα family has been identified in chicken (Thompson et al., 1998) and has been shown to mediate signalling of persephin via CRET (Enokido et al., 1998). A functional mammalian homologue encoding a mammalian persephin receptor has yet to be discovered.
The present inventors have surprisingly identified a further novel mammalian receptor of the GDNF family designated herein as GFRα-4. The DNA sequence has been cloned and a number of splice variants encoding the receptor have also been identified.